Isotopic labeling is a useful tool for rendering organic compounds easily identifiable in analytical and biochemical schemes. The isotopic label may be detected very sensitively, especially in the case of a radionuclide. By placing the isotopic label in a specific site in a molecule, it is possible to study reactions involving the molecule and detect and delineate reaction paths. Traditionally, isotopic hydrogen (e.g., tritium or deuterium) labeling has been limited by the unavailability of adequate deuterating/tritiating agents.
There are two fundamental techniques for introducing isotopic hydrogen into organic molecules. These are synthetic techniques and exchange techniques. Synthetic techniques, where tritium or deuterium is directly and specifically inserted, yield high tritium or deuterium abundance, but are limited by the chemistry required. In addition, the molecule being labeled may be changed, depending upon the severity of the synthetic reaction employed. Exchange techniques yield lower tritium or deuterium incorporation, often with the isotope being distributed over many sites on the molecule, but offer the advantage that they do not require separate synthetic steps and are less likely to disrupt the structure of the molecule being labeled.
Three common synthetic methods for incorporating activity levels of tritium into target molecules have been: (1) "hydrogenation" of the target molecule using tritium gas (T.sub.2), with a catalyst; (2) tritiodehalogenation; and (3) tritiomethylation with CT.sub.3 I. Each of these methods has been heavily employed in the art to achieve high levels of isotope incorporation, yet each involves reaction conditions that can affect the integrity of the target molecule. Conversely, the use of milder "tritium exchange" methods typically involves reduction in the level of tritium incorporated into the target molecule.
A fourth way of synthetically incorporating tritium into a target molecule which contains a reducible site is to contact the target molecule with a reducing agent which is capable of inserting one or more tritium atoms into the reducible site. This methodology essentially mimics reduction with hydrogen-inserting reducing agents.
Metal borohydrides such as LiAlH.sub.4 and NaBH.sub.4 are widely used mild reducing agents. In contrast, lithium trialkylborohydride (superhydride) (Brown, H. C. et al., (1980) J. Org. Chem. 45:1-12) is known to be a highly reactive nucleophilic reducing agent, and is now commonly used in organic synthesis (Brown, H.C. et al., (1979) Aldrichimica Acta 12:3-11). This reagent is capable of reducing esters, hindered alkyl halides (Brown, H.C. et al., (1973) J. Am. Chem. Soc. 95: 1669-1671) and toluene-p-sulphonates, in addition to exhibiting great sterioselectivity and steriospecificity, as in the reduction of epoxides. More hindered trialkylborohydrides (such as lithium or potassium tri-sec-butyl borohydride; known as L-selectride and K-selectride) exhibit even more steric control, as in the reduction of cyclic ketones (Fortunator, J. M. et al., (1975) J. Org. Chem. 41: 2194-2200). These remarkable hydride reducing agents are generally synthesized by reaction of the appropriate alkylborane with a metal hydride (Brown, H. C. et al (1980) J. Org. Chem. 45:1-12). It is clear that the ability to produce metal deuterides and tritides with high deuterium/tritium content would give access to a large number of deuteriated/tritiated reducing agents for chemoselective, regioselective and stereoselective labeling sequences, and allow high level deuterium/tritium incorporation through established synthetic routes with these highly reactive and selective reagents.
The utility of supertritide has been demonstrated (Hegde, S. et al., (1983) J. Chem. Soc. Chem. Commun., 1484-1485) by the reduction of acids, aldehydes, toluene-p-sulphonates and epoxides, but these reactions were conducted with supertritide of specific activities in the mCi/mmol range (100's of MBq/mmol). Later work (Coates, R. M. et al., (1986) Synthesis and Applications of Isotopically Labeled Compounds (Proc. 2nd Int. Symp.), 207-212) reported the synthesis of chiral methyl groups, starting with supertritide at approximately 3 Ci/mmol. This is still a factor of 10 below the theoretical maximum (one tritium atom per molecule gives a specific activity 28.72 Ci/mmol or 1063 GBq/mmol) and consequently this tritiation reagent has not been applied in those types of reactions where it is used in general chemistry. The same general statements are true for the availability and utility of LiAlT.sub.4. At this time, both LiAlD.sub.4 and LiEt.sub.3 BD are available commercially.
Although the complex hydrides are very useful reagents, the preparation of the initial metal hydrides has been problematic, especially where radioisotopes are involved. Metal hydrides may be prepared from the respective elements: e.g. atomic hydrogen produced in a glow discharge tube was found to rapidly react with various alkali metals, vacuum condensed as thin films on the reaction tube walls, to form metal hydrides (Ferrell, E. et al., (1934) J. Chem. Soc. 7-8). Other means of producing atomic hydrogen (or tritons) include dissociation of molecular hydrogen (tritium) by microwave discharge activation (Cao, G. Y. et al., (1984) Trans. Am. Nucl. Soc. 45:18-19) or on the surface of a hot tungsten wire (Moser, H. C. et al., (1962) J. Chem. Phys. 66:2272-2273). The latter two methods offer the advantages of being less limiting in scale and the option of exchange of tritons with LiH, thereby avoiding the use of liquid lithium. Tritide synthesis on a large scale has also been reported under conditions of high temperature and pressure, where lithium tritide was synthesized at 98% purity in an iron crucible at 750.degree. C., in the presence of three atmospheres of tritium gas (Bowman, R. C. et al., (1988) J. Nucl. Materials 154:318-331). The severe conditions and need for excessive tritium in this procedure make this option less attractive than the others outlined above, and only usable by the nuclear/fusion industries.
One other problem lies in the fact that once the hydride (deuteride or tritide) is formed by one of the above methods its chemical reactivity is reported to be low, and conversion into a complex hydride for use as a reducing reagent in organic synthesis may be sluggish. Hegde, S. et al., (1983) J. Chem. Soc. Chem. Commun., 1484-1485, reported that a typical reduction with such agents took several days at 150.degree. C.
The present invention is directed to the aforementioned problems. It provides a new method of in situ synthesis to generate a highly reactive alkali metal deuteride or tritide with a large proportion of its hydrogen present as deuterium or tritium from the respective deuterium or tritium gas. This material is then converted into a desirable highly selective labeling agent.